CROSS-REFERENCE TO RELATED APPLICATION
This application is a non-provisional application claiming benefit of the filing date of provisional application No. 61/365,550, filed Jul. 19, 2010, the entire contents of which are incorporated hereon by reference.
BACKGROUND OF THE INVENTION
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Alzheimer's disease (AD) is a currently incurable progressive chronic neurodegenerative disease characterized by severe cognitive decline, generally afflicting the elderly. AD is the 7th leading cause of all deaths in the US and 5th leading cause of death in Americans aged greater than 65. In 2009 12.5 billion hours of care was given to AD patients, at a cost of ˜$144 billion. By 2050 the numbers of people suffering from dementia is expected to approach 16 million in the US alone, absorbing hundreds of billions dollars in healthcare and related costs. By 2050 the incidence of AD is expected to approach nearly 1 million people/year with a total estimated prevalence of 11-16 million [2010 Alzheimer's disease facts and figures. Alzheimers Dement 6, 158-194 (2010).]. No treatment is available to prevent neurodegeneration. Existing symptomatic therapies temporarily slow cognitive decline. New therapies in clinical trials target various selective biochemical pathways hypothesized to be either necessary or sufficient for disease etiology. Since AD is really a ‘syndrome’ rather than a ‘disease’ multiple therapeutic modalities and approaches are likely to emerge from future clinical trial data. Drugs that promise to cure Alzheimer's disease are in clinical development but their effectiveness will rely on early diagnosis of the disease. It is presumed that therapeutic approaches that identify a novel serological biomarker for early detection of disease onset will be a key component of maximizing therapeutic efficacy, [Samgard, K., et al. Cerebrospinal fluid total tau as a marker of Alzheimer's disease intensity. Int J Geriatr Psychiatry 25:403-410 (2010)]. ‘Sporadic’ AD (99%) arises as a result of multiple factors, such as age, family history and others. Only 1% of cases are the result of established genetic variations. At present there is no universally accepted serum biomarker of early sporadic AD disease progression. The sensitivity of CSF biomarker measurements [Hu, W. T., et al. Novel CSF biomarkers for Alzheimer's disease and mild cognitive impairment. Acta Neuropathol (2010)] and brain imaging technologies [Petersen, R C., et al. Alzheimer's Disease Neuroimaging Initiative (ADNI): clinical characterization. Neurology 74:201-209 (2010)] are improving. However, the ability of these biomarkers to detect early stage disease has not been realized.
There are currently no universally accepted biomarkers in blood that correlate with disease progression in AD. Recent evaluation of a new kit assay designed to measure levels of various forms of Aβ protein in blood for possible use in early detection of Alzheimer's has been available for research use since the summer of 2007 (INNO-BIA plasma Aβ forms, Innogenetics, Gent Belgium). This test establishes an Aβ42/Aβ40 ratio that is lower in patients with a predisposition for developing mild cognitive impairment (MCI), which usually precedes AD. Unfortunately, peripheral Aβ measurements are subject to conflicting reports for a variety of reasons, [Cedazo-Minguez, A., and Winblad, B. Biomarkers for Alzheimer's disease and other forms of dementia: clinical needs, limitations and future aspects. Exp Gerontol 45, 5-14 (2010)]. A complex blood plasma molecular test for diagnosis of AD has recently been described, [Ray, S. et al. Classification and prediction of clinical Alzheimer's diagnosis based on plasma signaling proteins. Nat Med; 13: 1359-1362 (2007)] wherein 18 out of 120 signaling proteins were found with 90% accuracy to be predictive “markers” of AD. The statistical interpretations of these 18 signaling protein microarray proteins appears cumbersome and not readily converted to an easy to perform test.
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OF THE INVENTION
A laboratory method for screening, diagnosing, monitoring and/or staging early onset Alzheimer's disease which consists of mild cognitive impairment entails conducting a blood test after an oxidative exposure of serum to assay for the presence of an elevated level of redox-reactive autoantibodies.
A method of detecting or diagnosing early onset Alzheimer's disease in a subject, includes the steps of assaying an oxidized first blood sample from the subject to determine a baseline level of oxidized autoantibodies having selected specificities, treating a second longitudinal blood sample with an oxidizing agent and assaying the oxidized second sample to determine the level of autoantibodies having the selected specificities, and comparing the level of the autoantibodies in the first sample with the level of autoantibodies in the oxidized second sample, wherein an increase in the level of autoantibodies in the oxidized second sample as compared to the level of the oxidized first sample correlates with early onset Alzheimer's disease defined as mild cognitive impairment in said subject.
A blood serum biomarker for diagnosing, monitoring and/or staging early onset Alzheimer's disease defined as mild cognitively impairment comprising redox-reactive autoantibodies.
A kit for diagnosing, monitoring and/or staging early onset Alzheimer's disease defined as mildly cognitively impaired individuals includes a laboratory assay which can detect redox reactive autoantibodies before and after exposure to an oxidative agent.
BRIEF DESCRIPTION OF THE DRAWINGS
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FIG. 1. is a chart showing diagnostic treatment possibilities and evolution of irreversible dementia.
FIG. 2 shows a Classification And Regression Tree analysis (CART) of preliminary data results of applicant's R-RAA unmasked anti-PE ELISA in serum from MCI and normal donors (Values are OD units).
FIG. 3 shows hemin unmasked Redox Reactive Antibody Activity (R-RAA) in serum from normal age-matched controls and patients with mild cognitive impairment and Alzheimer's dementia.
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OF THE INVENTION
It would be desirable to have a method, biomarker and kit for early diagnosis of AD using a simple blood test. Identification of a robust serum biomarker for identifying early asymptomatic AD will be essential for maximal efficacy of future therapeutics currently in clinical development aimed at both halting disease progression and/or modifying the rate of cognitive decline. The sensitivity of CSF biomarkers and brain imaging technologies to stage AD disease progression are improving, but at present there are no inexpensive laboratory tests that are clinically useful to identify at-risk individuals for developing AD. The determination of diagnostic accuracy for novel AD biomarkers requires studies of samples of bodily fluids obtained longitudinally from individuals until AD can be confirmed at autopsy. Applicant can demonstrate a serum biomarker from anamnestic mild cognitively impaired (MCI) subjects that can discriminate, at high levels of significance, serum samples derived from MCI patients versus those from AD patients and/or normal age-matched controls. A serum biomarker for the detection and staging of AD is an ideal minimally-invasive technique that can be used in routine annual screening.
The basis of applicant's AD biomarker invention is based on the discovery of a novel family of autoantibodies present in serum that have their antigen recognition sites “masked.” Oxidative unmasking of these redox-reactive autoantibodies (R-RAA) in serum samples in vitro in the presence of their recognition epitopes quantitatively measures concentration of unmasked antibodies in serum using an ELISA format. Applicant's R-RAA technology has the potential for revolutionizing the medical community's ability to identify at-risk MCI individuals by assessing their increase in R-RAA over their base line values and their increased ELISA reactivity compared to AD and/or normal age-matched individuals.
Using patented technology, the identification and validation of a novel serum biomarker assay for the purpose of developing an in vitro diagnostic (IVD) was attempted. The initial requirements used to guide the design, format and implementation of the IVD were based on the following: (a) the 1998 Consensus Report [Consensus report of the working group on molecular and biochemical markers of Alzheimer's disease The Ronald and Nancy Reagan Research Institute of Alzheimer's Association and the National Institute on Aging Working Group. Neurobiol Aging; 19: 109-116 (1998)]. “An ideal marker should have greater than 80% sensitivity and specificity for excluding other dementias and neurodegenerative processes; it should be reliable, reproducible, non-invasive, easy to perform, and inexpensive” (b) The reagent components used in the assay must be suitably stable; and (c) must incorporate applicant's proprietary technology in order to justify development, validation and implementation costs. Previous work by the present inventor demonstrated potential by using 16 AD serum samples and 17 age-matched serum samples from volunteer blood donors. Each serum was tested before and after unmasking of the R-RAA with hemin. R-RAA's specificities for PS, PC, CL and PE were evaluated by using an in-house ELISA. Comparisons between the AD and normal populations revealed highly significant differences in R-RAA antiphosphatidylethanolamine (aPE), McIntyre, J A, Wagenknecht, D R, and Ramsey, C J. Redox-reactive antiphospholipid antibody differences between serum from Alzheimer's patients and age-matched controls. Autoimmunity, 42:646-52, (2009). In-sample Fisher's linear discriminate analysis found a sensitivity of 88% and a specificity of 94%. In-sample Classification and Regression Tree analysis (CART) found a sensitivity of 84% and a specificity of 100%. This study was the first to indicate that blood tests for R-RAA may be used as a laboratory criterion for an Alzheimer's diagnosis. These preliminary data are encouraging because it met the 1998 Consensus report “that an ideal marker should have greater than 80% sensitivity”. Additionally, reduced serum ethanolamine plasmalogen in late stages of AD [Wood, P. L., Mankidy, R., Ritchie, S., Heath, D., Wood, J. A., Flax, J., and Goodenowe, D. B. Circulating plasmalogen levels and Alzheimer Disease Assessment Scale-Cognitive scores in Alzheimer patients. J Psychiatry Neurosci; 35:59-62 (2010)] indicate that serum biomarkers associated with AD are encouraging.
Taking a small sample of the patient's blood does not pose the problems or limitations for routine annual AD screening in the primary care physician's office encountered with sampling CSF by lumbar puncture. It is for this reason that identifying a reliable blood biomarker that strongly correlates with early neurodegenerative disease progression will revolutionize screening of individuals at risk for developing AD just as routine blood lipid panel testing has revolutionized treatment of the hyperlipidemias. Given the expected escalating incidence and prevalence of AD with an aging population as the 21st century unfolds, it is easy to see why routine annual screening with a biomarker for AD would be highly desirable. Indeed one has only to see how Prostate Specific Antigen (PSA) screening has gained acceptance as a routine test, not because absolute PSA levels in a given patient are necessarily predictive, but a relative increase or trend over several years of single patient's PSA values can be a powerful early warning sign for development of prostate cancer warranting further more invasive biopsy evaluation. It is this added power of longitudinal sampling data from individuals that can substantially increase the predictive sensitivity of the biomarker. For these reasons the identification of a serum biomarker that robustly correlates with MCI in early AD has the potential to also be measurable in the late asymptomatic phase of the disease, where therapeutic intervention has the potential of reversing disease progression. The identification, validation and commercialization of such a serum biomarker for identifying asymptomatic at-risk individuals during a routine annual physical exam will be potentially ‘disruptive’ technology that must be quickly and expeditiously evaluated for implementation.
Identification of a robust biomarker for identifying early asymptomatic AD will be essential for future therapeutic interventions aimed at halting disease progression rather than simply modifying the rate of cognitive decline. With sporadic AD an individual is unlikely to become aware of mild cognitive impairment (MCI) until fulminate disease progression is established. As shown in FIG. 1, which shows diagnostic treatment possibilities and evolution of irreversible dementia, this is precisely the stage of AD where patients invariably compensate for cognitive problems and are often in denial of their symptoms to family and to their primary care physician. The ‘grayness’ scale cartoon at the top of the figure denotes the ‘probability’ of disease reversal by therapeutics. The ‘grayness scale’ cartoon in the middle of the figure represents a theoretical time window where therapeutics currently in clinical trials may afford the possibility for complete reversal of disease, or at least halting disease progression. (Figure modified from Cedazo-Minguez, A., and Winblad, B. Biomarkers for Alzheimer's disease and other forms of dementia: clinical needs, limitations and future aspects. Exp Gerontol 45, 5-14 (2010))
For this reason individuals are unlikely to voluntarily seek CSF biomarker assessment for AD diagnosis by requesting a lumbar puncture procedure. To really address the escalating incidence and prevalence of AD the discovery of a serological biomarker that could accurately detect early AD would solve this problem. If treatment options became available that arrest the disease when caught early, the general public, physicians and insurance companies alike would demand annual blood sample screening for AD to ward off the terrible consequences of living with the disease and its associated devastating financial and social costs. The 1998 Consensus Report of the Working Group on Molecular and Biochemical Markers of AD determined that an ideal marker should have a greater than 80% sensitivity and specificity for excluding other dementias and neurodegenerative processes, and it should be reliable, reproducible, non-invasive, easy to perform, and inexpensive [Consensus report of the working group on molecular and biochemical markers of Alzheimer's disease The Ronald and Nancy Reagan Research Institute of Alzheimer's Association and the National Institute on Aging Working Group. Neurobiol Aging 19:109-116 (1998)].
The present inventor has previously reported the discovery that blood and other bodily fluids from normal individuals contain a significant number of antibodies, that, when treated with an oxidizing agent, become capable of binding self antigens. See, for example, the following publications:
McIntyre, J A. “The appearance and disappearance of antiphospholipid antibodies subsequent to oxidation-reduction reactions.” Thromb. Res. 2004; 114:579-87.
McIntyre, J A, Wagenknecht, D R, & Faulk, W P. “Autoantibodies unmasked by redox reactions.” J. Autoimmun 2005; 24:311-17.
McIntyre, J A, Wagenknecht, D R, & Faulk, W P. “Redox-reactive autoantibodies: Detection and physiological relevance. Autoimm. Rev. 2006; 5:76-83. and U.S Patent Application Publication No. 2005/0101016 A1.
McIntyre, J A & Faulk W P. Redox-reactive autoantibodies: biochemistry, characterization, and specificities. Clin Rev Allergy Immunol 37, 49-54 (2009).
McIntyre, J A, Wagenknecht, D R, and Ramsey, C J. Redox-reactive antiphospholipid antibody differences between serum from Alzheimer's patients and age-matched controls. Autoimmunity, 42: 646-52 (2009).
The entire contents of each of these publications are incorporated herein by reference.
Such autoantibodies may be detected by treating the blood or other bodily fluid with an oxidizing agent and then using a screening assay to detect antibodies that bind a self antigen. It has been found that such autoantibodies are present in blood or other bodily fluids in a wide variety of isotypes and specificities. It has also been found that autoantibodies can be detected in a purified or fractionated immunoglobulin composition that has been treated with oxidizing agents. Since the autoantibodies are not detected above a minimal baseline in blood or other bodily fluids from normal individuals or in immunoglobulin compositions pooled from normal individuals in the absence of an oxidation step, antibodies or autoantibodies having this property are referred to herein as “masked” antibodies or “masked” autoantibodies, and the process of treating blood or other bodily fluids or immunoglobulin preparations with oxidizing conditions is referred to herein as “unmasking” the masked antibodies or autoantibodies. Antibodies having the property of becoming masked or unmasked, depending on oxidation-reduction conditions may also be referred to herein as “redox-reactive autoantibodies” (R-RAA).
The present inventor set out to develop a novel serological biomarker as a diagnostic tool with sufficient sensitivity and predictability to be clinically useful to identify asymptomatic individuals at-risk for developing early stage AD. R-RAA technology is based on the identification of disease-specific serum autoantibodies as a biomarker for disease progression [McIntyre, J. A., Wagenknecht, D. R., and Ramsey, C. J. Redox-reactive antiphospholipid antibody differences between serum from Alzheimer's patients and age-matched controls. Autoimmunity, 42:646-52 (2009)]. The present inventor has developed proprietary methods to ‘unmask’ these autoantibodies in serum samples in vitro and has further identified autoantibodies reactive to AD relevant epitopes.
To further the AD studies, a pilot phase 1 study consisted of blinded sets of six samples from MCI, AD and cognitively normal donors (ND) were provided by the AD Neuroimaging Initiative (ADNI). These 18 coded frozen serum samples from ADNI were used for ‘blinded’ analysis of R-RAA aPE measurements. A totally unexpected observation emerged upon decoding the sample groups. In this study, as shown in the following Table and in FIG. 2, the MCI individuals had markedly elevated serum R-RAA aPE compared to NC (P=0.0003, FIG. 2).
t-Test: Two-Sample Assuming Unequal Variances